Bronchoalveolar lavage fluid IMMY Sona Aspergillus lateral-flow assay for the diagnosis of invasive pulmonary aspergillosis: a prospective, real life evaluation.

Prompt and reliable diagnosis of invasive pulmonary aspergillosis (IPA) is essential for early initiation of antifungal therapy. We evaluated bronchoalveolar lavage (BAL) fluid IMMY Sona Aspergillus lateral-flow assay (IMMY LFA) in 92 individuals with suspected pulmonary infection. Sensitivity and specificity (vs. host factor but no IPA) of BAL IMMY LFA for diagnosis of IPA in individuals with any European Organisation for Research and Treatment of Cancer-defied "host factor" were 67% and 85%, respectively. Performance appeared better in individuals with renal transplantation (100%, 100%), compared to those with hematological malignancy and/or allogenic stem cell transplantation (70%, 78%). We found BAL IMMY LFA to be a convenient and useful addition to our diagnostic armory for IPA. LAY ABSTRACT: We evaluated a new test for diagnosing invasive pulmonary aspergillosis from bronchoscopy samples. We tested 92 people and found that it was 67% sensitive and 85% specific (compared to diagnosis according to a set of internationally recognised criteria). We found this test convenient and useful.

Posaconazole MIC Distributions for Aspergillus fumigatus Species Complex by Four Methods: Impact of cyp51A Mutations on Estimation of Epidemiological Cutoff Values.

Estimating epidemiological cutoff endpoints (ECVs/ECOFFS) may be hindered by the overlap of MICs for mutant and nonmutant strains (strains harboring or not harboring mutations, respectively). Posaconazole MIC distributions for the Aspergillus fumigatus species complex were collected from 26 laboratories (in Australia, Canada, Europe, India, South and North America, and Taiwan) and published studies. Distributions that fulfilled CLSI criteria were pooled and ECVs were estimated. The sensitivity of three ECV analytical techniques (the ECOFFinder, normalized resistance interpretation [NRI], derivatization methods) to the inclusion of MICs for mutants was examined for three susceptibility testing methods (the CLSI, EUCAST, and Etest methods). The totals of posaconazole MICs for nonmutant isolates (isolates with no known cyp51A mutations) and mutant A. fumigatus isolates were as follows: by the CLSI method, 2,223 and 274, respectively; by the EUCAST method, 556 and 52, respectively; and by Etest, 1,365 and 29, respectively. MICs for 381 isolates with unknown mutational status were also evaluated with the Sensititre YeastOne system (SYO). We observed an overlap in posaconazole MICs among nonmutants and cyp51A mutants. At the commonly chosen percentage of the modeled wild-type population (97.5%), almost all ECVs remained the same when the MICs for nonmutant and mutant distributions were merged: ECOFFinder ECVs, 0.5 µg/ml for the CLSI method and 0.25 µg/ml for the EUCAST method and Etest; NRI ECVs, 0.5 µg/ml for all three methods. However, the ECOFFinder ECV for 95% of the nonmutant population by the CLSI method was 0.25 µg/ml. The tentative ECOFFinder ECV with SYO was 0.06 µg/ml (data from 3/8 laboratories). Derivatization ECVs with or without mutant inclusion were either 0.25 µg/ml (CLSI, EUCAST, Etest) or 0.06 µg/ml (SYO). It appears that ECV analytical techniques may not be vulnerable to overlap between presumptive wild-type isolates and cyp51A mutants when up to 11.6% of the estimated wild-type population includes mutants.

Multicenter, International Study of MIC/MEC Distributions for Definition of Epidemiological Cutoff Values for Sporothrix Species Identified by Molecular Methods.

Clinical and Laboratory Standards Institute (CLSI) conditions for testing the susceptibilities of pathogenic Sporothrix species to antifungal agents are based on a collaborative study that evaluated five clinically relevant isolates of Sporothrixschenckii sensu lato and some antifungal agents. With the advent of molecular identification, there are two basic needs: to confirm the suitability of these testing conditions for all agents and Sporothrix species and to establish species-specific epidemiologic cutoff values (ECVs) or breakpoints (BPs) for the species. We collected available CLSI MICs/minimal effective concentrations (MECs) of amphotericin B, five triazoles, terbinafine, flucytosine, and caspofungin for 301 Sporothrix schenckii sensu stricto, 486 S. brasiliensis, 75 S. globosa, and 13 S. mexicana molecularly identified isolates. Data were obtained in 17 independent laboratories (Australia, Europe, India, South Africa, and South and North America) using conidial inoculum suspensions and 48 to 72 h of incubation at 35°C. Sufficient and suitable data (modal MICs within 2-fold concentrations) allowed the proposal of the following ECVs for S. schenckii and S. brasiliensis, respectively: amphotericin B, 4 and 4 µg/ml; itraconazole, 2 and 2 µg/ml; posaconazole, 2 and 2 µg/ml; and voriconazole, 64 and 32 µg/ml. Ketoconazole and terbinafine ECVs for S. brasiliensis were 2 and 0.12 µg/ml, respectively. Insufficient or unsuitable data precluded the calculation of ketoconazole and terbinafine (or any other antifungal agent) ECVs for S. schenckii, as well as ECVs for S. globosa and S. mexicana These ECVs could aid the clinician in identifying potentially resistant isolates (non-wild type) less likely to respond to therapy.

Operative and survival outcomes in a series of 100 consecutive cases of robot-assisted transhiatal esophagectomies.

Robotic-assisted transhiatal esophagectomy (RATE) is a technically complex procedure with potential for improved postoperative outcomes. In this report, we describe our experience with RATE in a large case series. A retrospective review was conducted to collect clinical, outcomes, and survival data for 100 consecutive patients with esophageal cancer (n = 98) and benign (n = 2) conditions undergoing RATE between March 2007 and December 2014. Progression-free (PFS) and overall (OS) survival were estimated using the Kaplan-Meier curves with comparisons by log-rank tests. Median operative time and estimated blood loss were 264 minutes and 75 mL, respectively. Median intensive care unit stay was 1 day and median length of hospital stay was 8 days. Postoperative complications commonly observed were nonmalignant pleural effusion (38%) and recurrent laryngeal nerve injury (33%); 30 day mortality rate was 2%. Median number of lymph nodes removed during RATE was 17 and R0 resection was achieved in 97.8% patients. At the end of the median follow-up period of 27.7 months, median PFS was 41 months and median OS was 54 months. 1-year and 3-year PFS rates were 82% (95% CI, 75%-89%) and 53% (95% CI, 42%-62%), respectively, and OS rates were 95% (95% CI, 91%-99%) and 57% (95% CI, 46%-67%). In our experience, RATE is an effective and safe oncologic surgical procedure in a carefully selected group of patients with acceptable operative time, minimal blood loss, standard postoperative morbidity and adequate PFS and OS profiles.

Post-mortem assessment of the short and long-term effects of the trophic factor neurturin in patients with α-synucleinopathies.

Substantial interest persists for developing neurotrophic factors to treat neurodegenerative diseases. At the same time, significant progress has been made in implementing gene therapy as a means to provide long-term expression of bioactive neurotrophic factors to targeted sites in the brain. Nonetheless, to date, no double-blind clinical trial has achieved positive results on its primary endpoint despite robust benefits achieved in animal models. A major issue with advancing the field is the paucity of information regarding the expression and effects of neurotrophic factors in human neurodegenerative brain, relative to the well-characterized responses in animal models. To help fill this information void, we examined post-mortem brain tissue from four patients with nigrostriatal degeneration who had participated in clinical trials testing gene delivery of neurturin to the putamen of patients. Each had died of unrelated causes ranging from 1.5-to-3-months (2 Parkinson's disease patients), to 4+-years (1 Parkinson's disease and 1 multiple-system atrophy-parkinsonian type patient) following gene therapy. Quantitative and immunohistochemical evaluation of neurturin, alpha-synuclein, tyrosine hydroxylase (TH) and an oligodendroglia marker (Olig 2) were performed in each brain. Comparable volumes-of-expression of neurturin were seen in the putamen in all cases (~15-22%; mean=18.5%). TH-signal in the putamen was extremely sparse in the shorter-term cases. A 6-fold increase was seen in longer-term cases, but was far less than achieved in animal models of nigrostriatal degeneration with similar or even far less NRTN exposure. Less than 1% of substantia nigra (SN) neurons stained for neurturin in the shorter-term cases. A 15-fold increase was seen in the longer-term cases, but neurturin was still only detected in ~5% of nigral cells. These data provide unique insight into the functional status of advanced, chronic nigrostriatal degeneration in human brain and the response of these neurons to neurotrophic factor stimulation. They demonstrate mild but persistent expression of gene-mediated neurturin over 4-years, with an apparent, time-related amplification of its transport and biological effects, albeit quite weak, and provide unique information to help plan and design future trials.

Sarcocystis fayeri-Induced Granulomatous and Eosinophilic Myositis in 2 Related Horses.

This report describes 2 genetically related paint mares, case Nos. 1 and 2, presented to the Oklahoma State University Boren Veterinary Medical Teaching Hospital for chronic weight loss and abnormal gait, respectively. Notable findings in both cases included marked persistent eosinophilia and multiple intramuscular lateral thoracic masses. Histologic examination of masses revealed eosinophilic, centrally necrotic granulomas and marked eosinophilic myositis. Granulomas in case No. 1 also contained intralesional Sarcocystis sp material, and adjacent muscle fibers contained intact protozoal cysts. Case No. 1 developed severe refractory muscle pain and recurrent esophageal dysphagia. At necropsy, disseminated, grossly visible granulomas were present throughout all examined striated muscles. Nested polymerase chain reaction of the 18S rRNA gene revealed >99% homology with Sarcocystis fayeri. Sarcocystis spp are apicomplexan protozoa that infect striated muscle of many omnivorous species, typically without inciting clinical disease. Sarcocystosis should be considered a rare cause of granulomatous eosinophilic myositis and choke in horses.

Robot-assisted transhiatal esophagectomy: a 3-year single-center experience.

Minimally invasive esophagectomy has emerged as an important procedure for disease management in esophageal cancer (EC) with clear margin status, less morbidity, and shorter hospital stays compared with open procedures. The experience with transhiatal approach robotic esophagectomy (RE) for dissection of thoracic esophagus and associated morbidity is described here. Between March 2007 and November 2010, 40 patients with resectable esophageal indications underwent transhiatal RE at the institute. Clinical data for all patients were collected prospectively. Of 40 patients undergoing RE, one patient had an extensive benign stricture, one had high-grade dysplasia, and 38 had EC. Five patients were converted from robotic to open. Median operative time and estimated blood loss were 311 minutes and 97.2 mL, respectively. Median intensive care unit stay was 1 day (range, 0-16), and median length of hospital stay was 9 days (range, 6-36). Postoperative complications frequently observed were anastomotic stricture (n= 27), recurrent laryngeal nerve paresis (n= 14), anastomotic leak (n= 10), pneumonia (n= 8), and pleural effusion (n= 18). Incidence rates of laryngeal nerve paresis (35%) and leak rate (25%) were somewhat higher in comparison with that reported in literature. However, all vocal cord injuries were temporary, and all leaks healed following opening of the cervical incision and drainage. None of the patients died in the hospital, and 30-day mortality was 2.5% (1/40). Median number of lymph nodes removed was 20 (range, 3-38). In 33 patients with known lymph node locations, median of four (range, 0-12) nodes was obtained from the mediastinum, and median of 15 (range, 1-26) was obtained from the abdomen. R0 resection was achieved in 94.7% of patients. At the end of the follow-up period, 25 patients were alive, 13 were deceased, and 2 patients were lost to follow-up. For patients with EC, median disease-free survival was 20 months (range, 3-45). Transhiatal RE, by experience, is a feasible albeit evolving oncologic operation with low hospital mortality. The benefits include minimally invasive mediastinal dissection without thoracotomy or thoracoscopy. A reasonable operative time with minimal blood loss and postoperative morbidity can be achieved, in spite of the technically demanding nature of the procedure. Broader use of this technology in a setting of high-volume comprehensive surgical programs will almost certainly reduce the complication rates. Robotic tanshiatal esophagectomy with the elimination of a thoracic approach should be considered an option for the appropriate patient population in a comprehensive esophageal program.

Examining new phylogenetic markers to uncover the evolutionary history of early-diverging fungi: comparing MCM7, TSR1 and rRNA genes for single- and multi-gene analyses of the Kickxellomycotina.

The recently recognised protein-coding genes MCM7 and TSR1 have shown significant promise for phylogenetic resolution within the Ascomycota and Basidiomycota, but have remained unexamined within other fungal groups (except for Mucorales). We designed and tested primers to amplify these genes across early-diverging fungal clades, with emphasis on the Kickxellomycotina, zygomycetous fungi with characteristic flared septal walls forming pores with lenticular plugs. Phylogenetic tree resolution and congruence with MCM7 and TSR1 were compared against those inferred with nuclear small (SSU) and large subunit (LSU) rRNA genes. We also combined MCM7 and TSR1 data with the rDNA data to create 3- and 4-gene trees of the Kickxellomycotina that help to resolve evolutionary relationships among and within the core clades of this subphylum. Phylogenetic inference suggests that Barbatospora, Orphella, Ramicandelaber and Spiromyces may represent unique lineages. It is suggested that these markers may be more broadly useful for phylogenetic studies among other groups of early-diverging fungi.

Isolation of the fungus Geosmithia argillacea in sputum of people with cystic fibrosis.

We report the repeated isolation of the fungus Geosmithia argillacea from sputum samples of people with cystic fibrosis. Identification was based on morphology and DNA sequence analysis. Isolation of G. argillacea did not appear to be associated with clinical deterioration. The pathogenic potential of G. argillacea is discussed.

Using routine blood parameters to anticipate clinical outcomes in invasive aspergillosis.

OBJECTIVE: In invasive aspergillosis (IA), monitoring response to antifungal treatment is challenging. We aimed to explore if routine blood parameters help to anticipate outcomes following IA. METHODS: Post hoc secondary analysis of two multicenter randomized trials was performed. The Global Comparative Aspergillosis Study (GCA, n = 123) and the Combination Antifungal Study (CAS, n = 251) constituted the discovery and validation cohorts respectively. The outcome measures were response to treatment and survival to 12 weeks. Interval platelet, galactomannan index (GMI) and C-reactive protein (CRP) levels prior and during antifungal treatment were analysed using logistic regression, Kaplan-Meier survival and receiver operating characteristic (ROC) analyses. RESULTS: The 12-week survival was 70.7% and 63.7% for the GCA and CAS cohorts respectively. In the GCA cohort, every 10 × 109/L platelet count increase at week 2 and 4 improved 12-week survival odds by 6-18% (odds ratio (OR) 1.06-1.18, 95% confidence interval (CI) 1.02-1.33). Survival odds also improved 13% with every 10 mg/dL CRP drop at week 1 and 2 (OR 0.87, 95% CI 0.78-0.97). In the CAS cohort, week 2 platelet count was also associated with 12-week survival with 10% improved odds for every 10 × 109/L platelet increase (OR, 1.10, 95% CI 1.04-1.15). A GMI drop of 0.1 unit was additionally found to increase the odds of treatment response by 3% at the baseline of week 0 (OR 0.97, 95% CI 0.95-0.99). Week 2 platelet and CRP levels performed better than GMI on ROC analyses for survival (area under ROC curve 0.76, 0.87 and 0.67 respectively). A baseline platelet count higher than 30 × 109/L clearly identified patients with >75% survival probability. CONCLUSIONS: Higher serial platelets were associated with overall survival while GMI trends were linked to IA treatment response. Routine and simple laboratory indices may aid follow-up of response in IA patients.

Analysis of Ret knockin mice reveals a critical role for IKKs, but not PI 3-K, in neurotrophic factor-induced survival of sympathetic neurons.

We analyzed the survival responses and downstream signaling elicited by GDNF on sympathetic neurons from different Ret knockin mice. Lack of tyrosine 1062, a multidocking site in Ret, completely prevented GDNF-mediated survival. Importantly, lack of tyrosine 981, although abrogating Akt phosphorylation, had no effect on neuronal survival, indicating that the PI 3-K/Akt pathway is not necessary for survival of sympathetic neurons. In contrast, silencing of B-Raf completely prevented not only GDNF-mediated but also NGF-mediated cell survival, independently of MEK-1/2. We identified IKKs as the main effectors of the protective effects of B-Raf. First, B-Raf interacted with and activated IKKs. Second, knockdown of IKKs reversed the protection afforded by a constitutively active form of B-Raf. Third, knockdown of IKKs prevented both NGF- and GDNF-mediated survival. In conclusion, our data delineate a novel survival pathway for sympathetic neurons linking B-Raf to IKKs, independently of both PI 3-K and MEK-1/2 pathways.

Wild-type MIC distribution and epidemiological cutoff values for Aspergillus fumigatus and three triazoles as determined by the Clinical and Laboratory Standards Institute broth microdilution methods.

Antifungal susceptibility testing of Aspergillus species has been standardized by both the Clinical and Laboratory Standards Institute (CLSI) and the European Committee on Antimicrobial Susceptibility Testing (EUCAST). Recent studies suggest the emergence of strains of Aspergillus fumigatus with acquired resistance to azoles. The mechanisms of resistance involve mutations in the cyp51A (sterol demethylase) gene, and patterns of azole cross-resistance have been linked to specific mutations. Studies using the EUCAST broth microdilution (BMD) method have defined wild-type (WT) MIC distributions, epidemiological cutoff values (ECVs), and cross-resistance among the azoles. We tested a collection of 637 clinical isolates of A. fumigatus for which itraconazole MICs were < or = 2 microg/ml against posaconazole and voriconazole using the CLSI BMD method. An ECV of < or = 1 microg/ml encompassed the WT population of A. fumigatus for itraconazole and voriconazole, whereas an ECV of < or = 0.25 microg/ml was established for posaconazole. Our results demonstrate that the WT distribution and ECVs for A. fumigatus and the mold-active triazoles were the same when determined by the CLSI or the EUCAST BMD method. A collection of 43 isolates for which itraconazole MICs fell outside of the ECV were used to assess cross-resistance. Cross-resistance between itraconazole and posaconazole was seen for 53.5% of the isolates, whereas cross-resistance between itraconazole and voriconazole was apparent in only 7% of the isolates. The establishment of the WT MIC distribution and ECVs for the azoles and A. fumigatus will be useful in resistance surveillance and is an important step toward the development of clinical breakpoints.

Characterization of the binding properties and retrograde axonal transport of a monoclonal antibody directed against the rat nerve growth factor receptor.

We have demonstrated in vitro and in vivo the specific binding of a monoclonal antibody to the rat nerve growth factor (NGF) receptor. Previous work had shown that this antibody, designated 192-IgG, does not compete with NGF for binding to the NGF receptor of PC12 cells, but instead interacts with the receptor to increase NGF binding to PC12 cells (Chandler, C. E., L. M. Parsons, M. Hosang, and E. M. Shooter, 1984, J. Biol. Chem., 259:6882-6889). In the present study, a solid-phase separation assay verified the specific formation of a ternary complex of 192-IgG, the NGF receptor, and NGF: 125I-labeled 192-IgG precipitated from solution only when incubated with both solubilized NGF receptor and NGF covalently linked to a solid phase (Sepharose 4B). Filtration assays using plasma membrane preparations of various tissues showed strict correlation of 125I-192-IgG and 125I-labeled NGF binding; only membranes obtained from superior cervical ganglion bound significant amounts of the monoclonal antibody and NGF. Injection of 125I-192-IgG into the rat anterior eye chamber led to accumulation of intact antibody molecules in the ipsilateral superior cervical ganglion, indicating retrograde axonal transport of 125I-192-IgG from the neuronal termini, located at the iris, to the cell bodies situated in the ganglion. The time course and saturation characteristics of 125I-192-IgG retrograde transport were very similar to those previously reported for 125I-NGF transport, indicating that 192-IgG can be internalized and transported by the same mechanisms as is NGF. Consistent with results of the in vitro binding assays, 192-IgG and NGF failed to compete for retrograde transport and were actually co-transported. Retrograde axonal transport of 192-IgG appears to be species specific, since 125I-192-IgG was transported in the rat, but not in mice, gerbils, hamsters, or guinea pigs. These results establish monoclonal antibody 192-IgG as a specific probe for the rat NGF receptor in vitro and in vivo.

Temporal analysis of events associated with programmed cell death (apoptosis) of sympathetic neurons deprived of nerve growth factor.

The time course of molecular events that accompany degeneration and death after nerve growth factor (NGF) deprivation and neuroprotection by NGF and other agents was examined in cultures of NGF-dependent neonatal rat sympathetic neurons and compared to death by apoptosis. Within 12 h after onset of NGF deprivation, glucose uptake, protein synthesis, and RNA synthesis fell precipitously followed by a moderate decrease of mitochondrial function. The molecular mechanisms underlying the NGF deprivation-induced decrease of protein synthesis and neuronal death were compared and found to be different, demonstrating that this decrease of protein synthesis is insufficient to cause death subsequently. After these early changes and during the onset of neuronal atrophy, inhibition of protein synthesis ceased to halt neuronal degeneration while readdition of NGF or a cAMP analogue remained neuroprotective for 6 h. This suggests a model in which a putative killer protein reaches lethal levels several hours before the neurons cease to respond to readdition of NGF with survival and become committed to die. Preceding loss of viability by 5 h and concurrent with commitment to die, the neuronal DNA fragmented into oligonucleosomes. The temporal and pharmacological characteristics of DNA fragmentation is consistent with DNA fragmentation being part of the mechanism that commits the neuron to die. The antimitotic and neurotoxin cytosine arabinoside induced DNA fragmentation in the presence of NGF, supporting previous evidence that it mimicked NGF deprivation-induced death closely. Thus trophic factor deprivation-induced death occurs by apoptosis and is an example of programmed cell death.

Inhibition of apoptotic signaling cascades causes loss of trophic factor dependence during neuronal maturation.

During development, neurons are acutely dependent on target-derived trophic factors for survival. This dependence on trophic support decreases dramatically with maturation in several neuronal populations, including sympathetic neurons. Analyses of nerve growth factor deprivation in immature and mature sympathetic neurons indicate that maturation aborts the cell death pathway at a point that is mechanistically indistinguishable from Bax deletion. However, neither the mRNA nor protein level of BAX changes with neuronal maturation. Therefore, BAX must be regulated posttranslationally in mature neurons. Nerve growth factor deprivation in immature sympathetic neurons induces two parallel processes: (a) a protein synthesis-dependent, caspase-independent translocation of BAX from the cytosol to mitochondria, followed by mitochondrial membrane integration and loss of cytochrome c; and (b) the development of competence-to-die, which requires neither macromolecular synthesis nor BAX expression. Activation of both signaling pathways is required for caspase activation and apoptosis in immature sympathetic neurons. In contrast, nerve growth factor withdrawal in mature sympathetic neurons did not induce the translocation of either BAX or cytochrome c. Moreover, mature neurons did not develop competence-to-die with cytoplasmic accumulation of cytochrome c. Therefore, inhibition of both BAX-dependent cytochrome c release and the development of competence-to-die contributed to the loss of trophic factor dependence associated with neuronal maturation.

Control of neuronal size homeostasis by trophic factor-mediated coupling of protein degradation to protein synthesis.

We demonstrate that NGF couples the rate of degradation of long-lived proteins in sympathetic neurons to the rate of protein synthesis. Inhibiting protein synthesis rate by a specific percentage caused an almost equivalent percentage reduction in the degradation rate of long-lived proteins, indicating nearly 1:1 coupling between the two processes. The rate of degradation of short-lived proteins was unaffected by suppressing protein synthesis. Included in the pool of proteins that had increased half-lives when protein synthesis was inhibited were actin and tubulin. Both of these proteins, which had half-lives of several days, exhibited no degradation over a 3-d period when protein synthesis was completely suppressed. The half-lives of seven other long-lived proteins were quantified and found to increase by 84-225% when protein synthesis was completely blocked. Degradation-synthesis coupling protected cells from protein loss during periods of decreased synthesis. The rate of protein synthesis greatly decreased and coupling between degradation and synthesis was lost after removal of NGF. Uncoupling resulted in net loss of cellular protein and somatic atrophy. We propose that coupling the rate of protein degradation to that of protein synthesis is a fundamental mechanism by which neurotrophic factors maintain homeostatic control of neuronal size and perhaps growth.

Caspase inhibition extends the commitment to neuronal death beyond cytochrome c release to the point of mitochondrial depolarization.

Nerve growth factor (NGF) deprivation induces a Bax-dependent, caspase-dependent programmed cell death in sympathetic neurons. We examined whether the release of cytochrome c was accompanied by the loss of mitochondrial membrane potential during sympathetic neuronal death. NGF- deprived, caspase inhibitor-treated mouse sympathetic neurons maintained mitochondrial membrane potential for 25-30 h after releasing cytochrome c. NGF- deprived sympathetic neurons became committed to die, as measured by the inability of cells to be rescued by NGF readdition, at the time of cytochrome c release. In the presence of caspase inhibitor, however, this commitment to death was extended beyond the point of cytochrome c release, but only up to the subsequent point of mitochondrial membrane potential loss. Caspase-9 deficiency also arrested NGF-deprived sympathetic neurons after release of cytochrome c, and permitted these neurons to be rescued with NGF readdition. Commitment to death in the NGF-deprived, caspase- 9-deficient sympathetic neurons was also coincident with the loss of mitochondrial membrane potential. Thus, caspase inhibition extended commitment to death in trophic factor-deprived sympathetic neurons and allowed recovery of neurons arrested after the loss of cytochrome c, but not beyond the subsequent loss of mitochondrial membrane potential.